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Stimulus‐Dependent Dynamics of P53 in Single Cells Molecular Systems Biology 7; Article number 488; doi:10.1038/msb.2011.20 Citation: Molecular Systems Biology 7:488 & 2011 EMBO and Macmillan Publishers Limited All rights reserved 1744-4292/11 www.molecularsystemsbiology.com REPORT Stimulus-dependent dynamics of p53 in single cells Eric Batchelor, Alexander Loewer, Caroline Mock and Galit Lahav* Department of Systems Biology, Harvard Medical School, Boston, MA, USA * Corresponding author. Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. Tel.: þ 1 617 432 5621; Fax: þ 1 617 432 5012; E-mail: [email protected] Received 30.8.10; accepted 7.3.11 Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input–output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to c-radiation. We further found that while c-triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input-sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus- dependent dynamics of p53, including excitability and input-duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network. Molecular Systems Biology 7: 488; published online 10 May 2011; doi:10.1038/msb.2011.20 Subject Categories: simulation and data analysis; signal transduction Keywords: DNA damage; dynamics; excitability; feedback; live-cell imaging This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial Share Alike 3.0 Unported License, which allows readers to alter, transform, or build upon the article and then distribute the resulting work under the same orsimilar license to this one. The work must be attributed back to the original author and commercial use is not permitted without specific permission. Introduction In addition, the p53 response to DSBs was shown to be excitable—a full p53 pulse is triggered by either a sustained or The tumor suppressor protein p53 is induced in response to a transient input (Batchelor et al, 2008; Loewer et al, 2010). many stress signals (Horn and Vousden, 2007) and activates The mechanism generating excitability in the p53 response to various stress-response programs including cell-cycle arrest, DSBs has not yet been determined. senescence, and apoptosis. An important unanswered ques- Here we show that different stresses trigger different tion is how can a single protein orchestrate such a complex temporal profiles of p53. We found that the p53 dynamical response to different inputs? Much attention in this regard has response to UV significantly differs from the response to DSBs, been focused around the hypothesis that different post- and we provide new mechanistic insights about the role of translational modifications of p53 lead to activation of feedback controlling specific features of p53 dynamics, such as different downstream gene programs (Brooks and Gu, 2003; excitability and input-strength dependency. Bode and Dong, 2004). However, another potential level of regulation, which has not been explored as much in this system, is the temporal dynamics of p53. The dynamical behavior of p53 has been extensively Results and discussion characterized in response to one particular form of DNA We first set out to determine whether a distinct form of DNA damage, double-strand breaks (DSBs) caused by g-radiation or damage leads to a series of undamped pulses as was previously the radiomimetic drug neocarzinostatin (NCS) (Shiloh et al, shown in response to DSBs (Lahav et al, 2004; Geva-Zatorsky 1983). DSBs trigger a series of p53 pulses with fixed amplitude et al, 2006; Batchelor et al, 2008). We chose UV light, which is and duration, independent of the damage dose, whereas the known to cross-link consecutive pyrimidine bases leading to number of pulses increases with higher damage (Lahav et al, the exposure of single-stranded DNA (ssDNA). Previous 2004; Geva-Zatorsky et al, 2006). It is not clear whether other studies showed that population-averaged analysis can mask types of DNA damage lead to similar dynamical behavior. true dynamical responses (Lahav et al, 2004; Tay et al, 2010); & 2011 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2011 1 Stimulus-dependent dynamics of p53 in single cells E Batchelor et al A B 100 ng/ml NCS 2 J/m2 UV 800 800 400 400 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 p53-Venus (AU) p53-Venus (AU) Time (h) Time (h) Time (h) Time (h) Time (h) Time (h) D C 400 ng/ml NCS 8 J/m2 UV 800 800 400 400 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 p53-Venus (AU) p53-Venus (AU) Time (h) Time (h) Time (h) Time (h) Time (h) Time (h) E F 2 2.2 100 ng/ml NCS 2 J/m UV First pulse 2.2 2 200 ng/ml NCS 4 J/m UV 6 J/m2UV 400 ng/ml NCS 1.8 1.8 8 J/m2UV 10 J/m2UV 1.4 1.4 1 1 Average of p53-Venus (AU) 0.6 Average of p53-Venus (AU) 0.6 012345 0 5 10 15 20 25 30 Time (h) Time (h) G 600 H 400 600 500 400 500 300 400 300 400 300 200 200 300 200 200 100 100 Duration (min) 100 Duration (min) 100 Amplitude (AU) 0 0 Amplitude (AU) 0 0 100 200 400 100 200 400 24 6810 246810 NCS (ng/ml) NCS (ng/ml) UV (J/m2) UV (J/m2) Figure 1 Stimulus-dependent dynamics of p53 in response to NCS (DSBs) and to UV. (A–D) Representative traces of MCF7 cells expressing p53-Venus exposed to 100 (A) or 400 ng/ml (C) of NCS or to 2 (B) or 8 J/m2 (D) UV at the indicated time following treatment. (E, F) p53-Venus levels averaged over the traces of cells in response to various levels of NCS and UV. Note that for NCS, the averaged response is shown only for the first p53 pulse, as synchrony between cells is lost in subsequent pulses. Each experiment includes between 60 and 110 cells. The mean trace was normalized to the mean p53-Venus of the first time point. (G, H) Quantification of the relative amplitude and full-width half-maximum duration of all p53 pulses in response to NCS (G) or the first pulse in response to UV (H). Error bars represent s.e.m. we therefore analyzed p53 dynamics in single cells using a pulses in response to UV might be independent of the extrinsic p53-Venus fusion (Batchelor et al, 2008). We found that in UV damage, and likely represent pulses resulting from intrinsic response to a single short burst of 2 J/m2 UV, p53 showed damage during normal proliferation as was recently shown pulses that were similar in amplitude and duration to the (Loewer et al, 2010). pulses observed in response to DSBs caused by NCS (Figure 1A The amplitude, duration, and frequency of individual p53 and B). However, the frequency of the pulses in response to UV pulses in response to DSBs are fixed and do not depend on the was lower and did not appear to be as regular as the pulses in damage dose (Lahav et al, 2004; Geva-Zatorsky et al, 2006; response to DSBs. Figure 1A, C, E, and G). To determine whether this is also the To determine whether there was a principal frequency of the case in response to UV, we irradiated cells with a broad range UV-induced pulses, we performed pitch detection as was of UV doses, each delivered in a single burst. We found that previously described (Geva-Zatorsky et al, 2006). In contrast higher UV doses increased both the amplitude and the to the DSB response that results in a principal period of 4–7 h duration of the p53 response (Figure 1B, D, F, and H). No (Geva-Zatorsky et al, 2006), a broad distribution of pitches was principal frequency was identified by pitch detection for any of determined for the UV response (Supplementary Figure S1), the UV doses (Supplementary Figure S1), indicating that there indicating that there was no dominant frequency of p53 pulses. is no periodicity in the p53 response to UV. This dose- The irregularity of the pulse frequency suggests that later dependent, single p53 pulse in response to UV is in stark 2 Molecular Systems Biology 2011 & 2011 EMBO and Macmillan Publishers Limited Stimulus-dependent dynamics of p53 in single cells E Batchelor et al A DNA DSBs B ssDNA (γ, NCS) (UV) ATM ATR p53 p53 Wip1 Mdm2 Wip1 Mdm2 C D Simulated 2.2 First pulse 2.2 UV dose 1.8 1.8 2 J/m2 UV 4 J/m2 UV 1.4 1.4 6 J/m2 UV 8 J/m2 UV Simulated Simulated 1 1 10 J/m2 UV p53 levels (AU) p53 levels (AU) 0.6 0.6 012 3450 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (h) Time (h) Time (h) EF10 Gy γ 2 J/m2 UV 8 J/m2 UV Time (h) 012345678910 Time (h) 01234560123456 Chk2-P(T68) Chk1-P(S317) (ATM activity) (ATR activity) Chk2 Chk1 Tubulin Tubulin G 10 Gy γ 8 J/m2 UV H 10 Gy γ 8 J/m2 UV Time (h) 0 0.5 1 2 3 4 0 0.5 1 2 3 4 Time (h) 0 0.5 1 2 3 4 0 0.5 1 2 3 4 Chk2-P(T68) Chk1-P(S317) (ATM activity) (ATR activity) Chk2 Chk1 Tubulin Tubulin 1.5 1.5 10 Gy γ 10 Gy γ 2 8 J/m2 UV 1 8 J/m UV 1 Chk2-P 0.5 Chk1-P 0.5 Normalized Normalized 0 0 04132 04132 Time (h) Time (h) Figure 2 A single feedback switches repeated, fixed p53 pulses into a single dose-dependent pulse.
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